Despite advances in treatment, congestive heart failure remains an important cause of mortality in Western countries. Heart failure affects 5 million individuals in the United States alone, and is characterized by a 5-year mortality rate of ˜50% (Levy et al., Long-term trends in the incidence of and survival with heart failure. N. Engl. J. Med., 347:1397-402, 2002). An important hallmark of heart failure is reduced myocardial contractility (Gwathmey et al., Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res., 61:70-76, 1987).
In healthy heart muscle, and other striated muscle, calcium-release channels on the sarcoplasmic reticulum (SR), including ryanodine receptors (RyRs), facilitate coupling of the action potential to a muscle cell's contraction (i.e., excitation-contraction (EC) coupling). Contraction is initiated when calcium (Ca2+) is released from the SR into the surrounding cytoplasm. In heart failure, contractile abnormalities result, in part, from alterations in the signaling cascade that allows the cardiac action potential to trigger contraction. In particular, in failing hearts, the amplitude of the whole-cell Ca2+ transient is decreased (Beuckelmann et al., Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circ., 85:1046-55, 1992; Gomez et al., Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science, 276:800-06, 1997), and the duration prolonged (Beuckelmann et al., Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circ., 85:1046-55, 1992).
Cardiac arrhythmia, a common feature of heart failure, results in many of the deaths associated with the disease. Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans, and represents a major cause of morbidity and mortality (Chugh et al., Epidemiology and natural history of atrial fibrillation: clinical implications. J. Am. Coll. Cardiol., 37:371-78, 2001; Falk, R. H., Atrial fibrillation. N. Engl. J. Med, 344:1067-78, 2001). Despite AF's clinical importance, the molecular mechanisms underlying this arrhythmia are poorly understood, and treatment options are limited.
It is well established that structural and electrical remodeling—including shortening of atrial refractoriness, loss of rate-related adaptation of refractoriness (Wijffels et al., Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation, 92:1954-68, 1995; Morillo et al., Chronic rapid atrial pacing: structural, functional, and electrophysiological characteristics of a new model of sustained atrial fibrillation. Circulation, 91:1588-95, 1995; Elvan et al., Pacing-induced chronic atrial fibrillation impairs sinus node function in dogs: electrophysiological remodeling. Circulation, 94:2953-60, 1996; Gaspo et al., Functional mechanisms underlying tachycardia-induced sustained atrial fibrillation in a chronic dog model. Circulation, 96:4027-35, 1997), and shortening of the wavelength of re-entrant wavelets—accompany sustained tachycardia (Rensma et al., Length of excitation wave and susceptibility to reentrant atrial arrhythmias in normal conscious dogs. Circ. Res., 62:395-410, 1988). This remodeling is likely important in the development, maintenance and progression of atrial fibrillation.
Previous studies suggest that calcium handling may play a role in electrical remodeling in atrial fibrillation (Sun et al., Cellular mechanisms of atrial contractile dysfunction caused by sustained atrial tachycardia. Circulation, 98:719-27, 1998; Goette et al., Electrical remodeling in atrial fibrillation: time course and mechanisms. Circulation, 94:2968-74, 1996; Daoud et al., Effect of verapamil and procainamide on atrial fibrillation-induced electrical remodeling in humans. Circulation, 96:1542-50, 1997; Yu et al., Tachycardia-induced change of atrial refractory period in humans: rate dependency and effects of antiarrhythmic drugs. Circulation, 97:2331-37, 1998; Leistad et al., Atrial contractile dysfunction after short-term atrial fibrillation is reduced by verapamil but increased by BAY K8644. Circulation, 93:1747-54, 1996; Tieleman et al., Verapamil reduces tachycardia-induced electrical remodeling of the atria. Circulation, 95:1945-53, 1997). However, regulation of RyR2 during atrial fibrillations has not previously been reported.
Approximately 50% of all patients with heart disease die from fatal cardiac arrhythmias. In some cases, a ventricular arrhythmia in the heart may be rapidly fatal—a phenomenon referred to as “sudden cardiac death” (SCD). Fatal ventricular arrhythmias (and SCD) may also occur in young, otherwise-healthy individuals who are not known to have structural heart disease. In fact, ventricular arrhythmia is the most common cause of sudden death in otherwise-healthy individuals.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disorder in individuals with structurally-normal hearts. It is characterized by stress-induced ventricular tachycardia—a lethal arrhythmia that may cause SCD. In subjects with CPVT, physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to SCD in the absence of detectable structural heart disease (Laitinen et al., Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation, 103:485-90, 2001; Leenhardt et al., Catecholaminergic polymorphic ventricular tachycardia in children: a 7-year follow-up of 21 patients. Circulation, 91:1512-19, 1995; Priori et al., Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation, 106:69-74, 2002; Priori et al., Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation, 103:196-200, 2001; Swan et al., Arrhythmic disorder mapped to chromosome 1q42-q43 causes malignant polymorphic ventricular tachycardia in structurally normal hearts. J. Am. Coll. Cardiol., 34:2035-42, 1999).
CPVT is predominantly inherited in an autosomal-dominant fashion.
Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. Linkage studies and direct sequencing have identified mutations in the human RyR2 gene, on chromosome 1q42-q43, in individuals with CPVT (Laitinen et al., Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation, 103:485-90, 2001; Priori et al., Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation, 103:196-200, 2001; Swan et al., Arrhythmic disorder mapped to chromosome 1q42-q43 causes malignant polymorphic ventricular tachycardia in structurally normal hearts. J. Am. Coll. Cardiol., 34:2035-42, 1999).
There are three types of ryanodine receptors, all of which are highly-related Ca2+ channels: RyR1, RyR2, and RyR3. RyR1 is found in skeletal muscle, RyR2 is found in the heart, and RyR3 is located in the brain. The type 2 ryanodine receptor (RyR2) is the major Ca2+-release channel required for EC coupling and muscle contraction in cardiac striated muscle.
RyR2 channels are packed into dense arrays in specialized regions of the SR that release intracellular stores of Ca2+, and thereby trigger muscle contraction (Marx et al., Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science, 281:818-21, 1998). During EC coupling, depolarization of the cardiac-muscle cell membrane, in phase zero of the action potential, activates voltage-gated Ca2+ channels. In turn, Ca2+ influx through these channels initiates Ca2+ release from the SR via RyR2, in a process known as Ca2+-induced Ca2+ release (Fabiato, A., Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol., 245:C1-C14, 1983; Nabauer et al., Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science, 244:800-03, 1989). The RyR2-mediated, Ca2+-induced Ca2+ release then activates the contractile proteins which are responsible for cardiac muscle contraction.
RyR2 is a protein complex comprising four 565,000-dalton RyR2 polypeptides in association with four 12,000-dalton FK506 binding proteins (FKBPs), specifically FKBP12.6 (calstabin). FKBPs are cis-trans peptidyl-prolyl isomerases that are widely expressed and serve a variety of cellular functions (Marks, A. R., Cellular functions of immunophilins. Physiol. Rev., 76:631-49, 1996). FKBP12 proteins are tightly bound to, and regulate the function of, the skeletal ryanodine receptor, RyR1 (Brillantes et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994; Jayaraman et al., FK506 binding protein associated with the calcium release channel (ryanodine receptor). J. Biol. Chem., 267:9474-77, 1992); the cardiac ryanodine receptor, RyR2 (Kaftan et al., Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels from cardiac muscle. Circ. Res., 78:990-97, 1996); a related intracellular Ca2+-release channel, known as the type 1 inositol 1,4,5-triphosphate receptor (IP3R1) (Cameron et al., FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain. J. Biol. Chem., 272:27582-88, 1997); and the type 1 transforming growth factor β (TGFβ) receptor (TβRI) (Chen et al., Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO J., 16:3866-76, 1997). FKBP12.6 binds to the RyR2 channel (one molecule per RyR2 subunit), stabilizes RyR2-channel function (Brillantes et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994), and facilitates coupled gating between neighboring RyR2 channels (Marx et al., Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science, 281:818-21, 1998), thereby preventing aberrant activation of the channel during the resting phase of the cardiac cycle.
Phosphorylation of cardiac RyR2 by protein kinase (PKA) is an important part of the “fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca2+ released for a given trigger (Marks, A. R., Cardiac intracellular calcium release channels: role in heart failure. Circ. Res., 87:8-11, 2000). This signaling pathway provides a mechanism by which activation of the sympathetic nervous system, in response to stress, results in increased cardiac output required to meet the metabolic demands of the stress responses. Upon binding of catecholamines, β1- and β2-adrenergic receptors activate adenylyl cyclase via a stimulatory G-protein, Gαs. Adenylyl cyclase increases intracellular cAMP levels, which activate the cAMP-dependent PKA. PKA phosphorylation of RyR2 increases the open probability of the channel by dissociating calstabin2 (FKBP12.6) from the channel complex. This, in turn, increases the sensitivity of RyR2 to Ca2+-dependent activation (Hain et al., Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. J. Biol. Chem., 270:2074-81, 1995; Valdivia et al., Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science, 267:1997-2000, 1995; Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000).
Failing hearts (e.g., in patients with heart failure and in animal models of heart failure) are characterized by a maladaptive response that includes chronic hyperadrenergic stimulation (Bristow et al., Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N. Engl. J. Med., 307:205-11, 1982). The pathogenic significance of this stimulation in heart failure is supported by therapeutic strategies that decrease beta-adrenergic stimulation and left ventricular myocardial wall stress, and potently reverse ventricular remodeling (Barbone et al., Comparison of right and left ventricular responses to left ventricular assist device support in patients with severe heart failure: a primary role of mechanical unloading underlying reverse remodeling. Circulation, 104:670-75, 2001; Eichhorn and Bristow, Medical therapy can improve the biological properties of the chronically failing heart. A new era in the treatment of heart failure. Circulation, 94:2285-96, 1996). In heart failure, chronic beta-adrenergic stimulation is associated with the activation of beta-adrenergic receptors in the heart, which, through coupling with G-proteins, activate adenylyl cyclase and thereby increase intracellular cAMP concentration. cAMP activates cAMP-dependent PKA, which has been shown to induce hyperphosphorylation of RyR2.
Thus, chronic heart failure is a chronic hyperadrenergic state (Chidsey et al., Augmentation of plasma norepinephrine response to exercise in patients with congestive heart failure. N. Engl. J. Med., 267:650, 1962) which results in several pathologic consequences, including PKA hyperphosphorylation of RyR2 (Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000).
The PKA hyperphosphorylation of RyR2 has been proposed as a factor contributing to depressed contractile function and arrhythmogenesis in heart failure (Marks et al., Progression of heart failure: is protein kinase a hyperphosphorylation of the ryanodine receptor a contributing factor? Circulation, 105:272-75, 2002; Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000). Consistent with this hypothesis, PKA hyperphosphorylation of RyR2 in failing hearts has been demonstrated in vivo, both in animal models and in patients with heart failure undergoing cardiac transplantation (Antos et al., Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circ. Res., 89:997-1004, 2001; Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000; Ono et al., Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca2+ release in heart failure. Cardiovasc. Res., 48:323-31, 2000; Reiken et al., Beta-adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure. Circulation, 104:2843-48, 2001; Semsarian et al., The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J. Clin. Invest., 109:1013-20, 2002; Yano et al., Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca2+ leak through ryanodine receptor in heart failure. Circulation, 102:2131-36, 2000).
In failing hearts, the hyperphosphorylation of RyR2 by PKA induces the dissociation of the regulatory FKBP12.6 subunit from the RyR2 channel (Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000). This causes marked changes in the biophysical properties of the RyR2 channel. Such changes are evidenced by increased open probability (Po), due to an increased sensitivity to Ca2+-dependent activation (Brillantes et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994; Kaftan et al., Effects of rapamycin on ryanodine receptor/Ca2+-release channels from cardiac muscle. Circ. Res., 78:990-97, 1996); destabilization of the channel, resulting in subconductance states; and impaired coupled gating of the channels, resulting in defective EC coupling and cardiac dysfunction (Marx et al., Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science, 281:818-21, 1998). Thus, PKA-hyperphosphorylated RyR2 is very sensitive to low-level Ca2+ stimulation, and this manifests itself as an SR Ca2+ leak through the hyperphosphorylated channel.
The maladaptive response to stress in heart failure results in depletion of FKBP12.6 from the channel macromolecular complex. This leads to a shift to the left in the sensitivity of RyR2 to Ca2+-induced Ca2+ release, resulting in channels that are more active at low-to-moderate [Ca2+] (Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000; Yamamoto et al., Abnormal Ca2+ release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure. Cardiovasc. Res., 44:146-55, 1999; Yano et al., Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca2+ leak through ryanodine receptor in heart failure. Circulation, 102:2131-36, 2000). Over time, the increased “leak” through RyR2 results in resetting of the SR Ca2+ content to a lower level, which in turn reduces EC coupling gain and contributes to impaired systolic contractility (Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000).
Additionally, a subpopulation of RyR2 that are particularly “leaky” can release SR Ca2+ during the resting phase of the cardiac cycle, diastole. This results in depolarizations of the cardiomyocyte membrane known as delayed after-depolarizations (DADs), which are known to trigger fatal ventricular cardiac arrhythmias (Wehrens et al., FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell, 113:829-40, 2003).
In structurally-normal hearts, a similar phenomenon may be at work.
Specifically, it is known that exercise and stress induce the release of catecholamines that activate beta-adrenergic receptors in the heart. Activation of the beta-adrenergic receptors leads to hyperphosphorylation of RyR2 channels. Evidence also suggests that the hyperphosphorylation of RyR2 resulting from beta-adrenergic-receptor activation renders mutated RyR2 channels more likely to open in the relaxation phase of the cardiac cycle, increasing the likelihood of arrhythmias.
Cardiac arrhythmias are known to be associated with SR Ca2+ leaks in structurally-normal hearts. In these cases, the most common mechanism for induction and maintenance of ventricular tachycardia is abnormal automaticity. One form of abnormal automaticity, known as triggered arrhythmia, is associated with aberrant release of SR Ca2+, which initiates DADs (Fozzard, H. A., Afterdepolarizations and triggered activity. Basic Res. Cardiol., 87:105-13, 1992; Wit and Rosen, Pathophysiologic mechanisms of cardiac arrhythmias. Am. Heart J, 106:798-811, 1983). DADs are abnormal depolarizations in cardiomyocytes that occur after repolarization of a cardiac action potential. The molecular basis for the abnormal SR Ca2+ release that results in DADs has not been fully elucidated. However, DADs are known to be blocked by ryanodine, providing evidence that RyR2 may play a key role in the pathogenesis of this aberrant Ca2+ release (Marban et al., Mechanisms of arrhythmogenic delayed and early afterdepolarizations in ferret ventricular muscle. J. Clin. Invest., 78:1185-92, 1986; Song and Belardinelli, ATP promotes development of afterdepolarizations and triggered activity in cardiac myocytes. Am. J. Physiol., 267:H2005-11, 1994).
In view of the foregoing, it is clear that leaks in RyR2 channels are associated with a number of pathological states—in both diseased hearts and structurally-normal hearts. Accordingly, methods to repair the leaks in RyR2 could prevent heart failure, and fatal arrhythmias and fibrillations, in millions of patients.
JTV-519 (4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine monohydrochloride; also known as k201 or ICP-Calstan 100), a derivative of 1,4-benzothiazepine, is a new modulator of calcium-ion channels. In addition to regulating Ca2+ levels in myocardial cells, JTV-519 also modulates the Na+ current and the inward-rectifier K+ current in guinea pig ventricular cells, and inhibits the delayed-rectifier K+ current in guinea pig atrial cells. Studies have shown that JTV-519 has a strong cardioprotective effect against catecholamine-induced myocardial injury, myocardial-injury-induced myofibrillar overcontraction, and ischemia/reperfusion injury. In experimental myofibrillar overcontraction models, JTV-519 demonstrated greater cardioprotective effects than propranolol, verapamil, and diltiazem. Experimental data also suggest that JTV-519 effectively prevents ventricular ischemia/reperfusion by reducing the level of intracellular Ca2+ overload in animal models.